Myocytes sense substrate stiffness - New materials for mechanism and application Poke your hamstring while sitting relaxed, and you immediately recognize that tissues are soft and not nearly so rigid as the polystyrene or glass culture dishes commonly used for cell studies in vitro. This R21's "'develop and explore" objectives focus on systematically testing, with new gel materials and/or micropattemed systems of controlled thickness, the hypothesis that myocytes sense matrix compliance. 'Hardening" of arteries and fibrotic scarring after myocardial infarction can influence, we believe, cell phenotype because of the physical properties of the matrix. In muscular dystrophies, rigidifying fibrosis occurs in some tissues (eg. diaphragm) that do not regenerate muscle despite the presence of regeneration-competent cells. In our initial studies in culture, both smooth muscle cells and skeletal muscle cells appear highly sensitive to substrate compliance and in some conditions more sensitive than the density of adhesive ligand (Engler et al, Biophys J 2004). For skeletal muscle, a very narrow range of matrix elasticity - in fact a tissue-like stiffness - is required for classical striation of acto-myosin. New material systems down to nanometers in thickness are needed for deeper insight, but we will also propose stepping back from the myoblasts and myotubes to examine whether stem cells capable of myocyte differentiation can be directed by a sensitivity to substrate compliance. Cardiomyocytes will be examined in terms of their ability to spread, undergo myofibrillogenesis and even beat rhythmically on various substrates. In parallel, we will assess the roles of protein expression (eg. cycloheximide and protein over-expression) and post-translational modification (eg. phosphorylation) in these responses, and we will measure cell adhesion to the matrices by a novel micromanipulation method already developed in the lab (Griffin et al, Biophys d 2004). New materials include (1) polyacrylamide gel supports that are only nanometers thick, (2) polyelectrolyte multilayers (PEM) made by molecular layering of polycations and polyanions, and (3) matrices impregnated with novel polymeric rod-micelles of controlled flexibility. In addition to trying to establish a 'common denominator' of compliance for myocyte responses to matrix, some of the new materials will also be tested for utility in application. These include testing pharmacological agents to over-ride compliance sensing; blebbistatin, for example, is a specific myosin-II inhibitor that will modulate contractility and perhaps allow undifferentiated cells to perceive rigid, 'fibrotic-like" substrates as soft. PEM coating of stents will also be examined for inhibiting the proliferative response of smooth muscle cells implicated in restenosis.